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The purpose of this article is to review recent experimental data from our laboratory on immune-neuroendocrine communications, whereby the following findings will be discussed: (i) Experiments using an experimental stress model in rats revealed different doses of a defined stressor to exert opposite effects on the in vitro reactivity of peripheral blood lymphocytes (PBL). Stress effects are also dependent on the tissue origin of lymphocytes, and they seem to be exclusively mediated by adrenal hormones. (ii) The balance between the sympathetic and parasympathetic nervous system may be critical in extrinsic immunoregulation: Experimentally induced hypercatecholaminemia in rats seem to protect lymphocytes from immunosuppressing effects of other, endogenous stress hormones, but causes suppression of PBL activation, if beta-receptors are blocked at the same time. Chronic cholinergic stimuli exert enhancing effects on the cells of the thymus. (iii) A defect in the immune-neuroendocrine crosstalk may contribute to the occurrence of forbidden immune reactions, as has been shown in spontaneous and experimentally induced autoimmune diseases in animals models. Recent data indicate that the parasympathetic nervous system is involved in the transmission of immune messages to the central nervous system.

In-vitro evidence suggests that DNA methylation may be involved in the development of forbidden immune responses that can result in autoimmune disease. In the present study we examined in-vivo effects of 5-azacytidine (5-azaC), a substance that inhibits DNA methylation, on the immune system and the occurrence of a spontaneous autoimmune disease in the chicken model. We found that (1) treatment of young normal chickens with 1.0 mg/kg 5-azaC on 7 consecutive days caused a rapid degeneration of the central lymphoid organs thymus and bursa; (2) this regimen with 5-azaC apparently inhibited B cell maturation, as the frequency of cytoplasmic Ig+ plasma cells in the bone marrow was found to be significantly reduced, whereas the total number of bone marrow cells was unchanged; and (3) a chronic low-dose (0.5 and 1.0 mg/kg) application of 5-azaC through 6 weeks was found to significantly enhance the spontaneous autoimmune thyroiditis in newly hatched chickens of the Cornell C strain, as determined by anti-thyroglobulin autoantibody titres and histological analysis of thyroid gland infiltration. The possible implications of these data for the generation of pathogenic autoimmune responses are discussed.

Thyroglobulin-binding lymphoid cells were identified in the spleen of Obese strain (DS) chickens by their capacity to form rosettes with thyroglobulin-coated chicken red blood cells. The nature of these cells was studied in inhibition experiments using turkey anti-chicken bursa or thymus cell sera and rabbit antisera specific for chicken Ig, gamma, mu, alpha, Fabgamma or Fcgamma. Spleen cells actively synthesizing surface receptors for thyroglobulin were identified as B cells and the receptors found to be complete IgM molecules. Normal T cells became thyroglobulin-rosette-forming cells via passive adsorption of thyroglobulin antibodies, a phenomenon which could be inhibited competitively by the addition of normal chicken serum to the incubation medium. Thyroglobulin antibodies passively adsorbed onto the surface of normal T cells also belong to the IgM class as verified both by inhibition experiments and studies employing IgM and IgG fractions of a high titered OS serum for the preincubation of the cell suspensions. Only preincubation with the IgM fraction of the anti-thyroglobulin antibodies resulted in the formation of significant numbers of passive rosette-forming cells.

The B-L region of the chicken major histocompatibility complex (MHC), the so-called B -locus, corresponds to the murine H-2 I-region. Using alloantibodies and monoclonal antibodies to B-L we analyzed: (a) the tissue distribution of B-L + cells, (b) the function of B-L + cells, and (c) the possible role of B-L + cells in the development of spontaneous autoimmune thyroiditis (SAT) in Obese strain (OS) chickens. The tissue distribution of B-L + cells in peripheral blood and various lymphoid and nonlymphoid organs corresponds to what is known for mammals. In the bursa of Fabricius most lymphoid cells and the dendritic cells carry the B-L antigen; B-L + thymic nurse cells (TNC) first appear on day 17 of embryonic life; chickens possess dendritic B-L + cells in the skin resembling mammlian Langerhans cells; in addition we found that the microglia is unequivocally B-L + . B-L + peripheral blood lymphocytes (PBL) were separated with a fluorescence-activated cell sorter. Ten percent of unstimulated PBL and 60% of phytohemagglutinin (PHA) stimulated T-cell blasts are B-L + . In graft-vs-host (GvH) assays B-L − cells were identified as the effector cells. These cells respond to PHA and concanavalin A (Con A), but not to pokeweed mitogen (PWM). B-L + cells cannot be stimulated by Con A and PHA, but respond to PWM. They possess only a very low activity in GvH assays which can be inhibited by anti-T-cell sera. In OS chickens B-L + /non-B/, non-T and B-L + T (blasts?) cells are found in the “first line” of mononuclear cell infiltration in the thyroid glands. Most interesting, thyroid epithelial cells—which are normally B-L − —become B-L + in the neighbourhood of B-L + infiltrating mononuclear cells. This observation may be of significance for autoantigen presentation and perpetuation in autoimmune thyroiditis. Finally, OS thymuses contain significantly less TNC than normal controls.

The mitogenic response to Con A and the production of T cell growth factor or interleukin 2 (IL 2) by splenic and peripheral blood lymphocytes of obese strain (OS) chickens with spontaneous autoimmune thyroiditis have been investigated. By using an optimized method with Con A-coated chicken erythrocytes (MRC), lymphocytes of OS chickens were found to exhibit significantly elevated mitogenic responses as compared with cells from either Normal White Leghorn chickens (NWL) or animals of the Cornell C-Strain (CS), from which the OS has originally been developed. This difference was observed throughout ontogeny up to 15 mo of age, and was associated with increased levels of IL 2 activity in the culture supernatants. The elevated responsiveness of OS T lymphocytes was also found to be manifested in the expression of receptors for IL 2, because Con A-stimulated lymphocytes of OS birds were significantly more effective than those from normal controls in absorbing IL 2 activity from conditioned media (CM) of stimulated spleen cells. High concentrations of CM were suppressive in IL 2 assays, signaling the presence of an inhibitory factor(s) in addition to IL 2. An additional indication for defective immunoregulation was that CM from OS lymphocyte cultures showed significantly less of this suppressive activity in comparison with CM of normal (NWL and CS) lymphocyte cultures. Finally, the spontaneous uptake of 125IUdR of embryonic and early post hatching OS spleen lymphocytes was consistently and significantly enhanced. This difference, however, in contrast to the one observed in Con A responses, was found to decrease with age. The data are discussed in view of the contradictory results concerning T cell functions reported for several autoimmune states in mammals.

Lymphocytes were isolated from the infiltrated thyroid glands of 2- to 5-wk-old Obese strain (OS) chickens with spontaneous autoimmune thyroiditis (SAT). Immunofluorescence analysis performed by using a panel of monoclonal and polyclonal antibodies revealed that 60% of thyroid infiltrating leukocytes (TIL) were mature T cells, a large portion of which seemed to be in an activated state bearing Ia-like antigens (10%) as well as a surface determinant associated with T cell activation (16%), i.e., possibly the receptor for interleukin 2 (IL 2). Furthermore, a relatively high plasma cell content (5%) was observed. TIL exhibited high proliferative responses to T cell mitogens (concanavalin A, phytohemagglutinin) and IL 2, but only weak responses to the B cell mitogen LPS from Salmonella typhimurium. When injected into newly hatched, MHC-identical, irradiated normal chickens, TIL induced both the production of autoantibodies and thyroid infiltration. Peripheral lymphocytes from spleen and blood and thymocytes from the same OS donors had no effect. Analysis of chemically (cyclophosphamide) bursectomized OS chickens suggested that an intact B cell system was not obligatory for the induction of SAT. TIL from these chickens consisted of 77% T cells and less than 1% B lymphocytes, yet were capable of inducing severe thyroid infiltration upon transfer into normal recipients. These findings emphasize the importance of the T cell system in the initiation of SAT.

Spontaneous autoimmune thyroiditis of OS chickens is associated with a marked hyperreactivity of the T cell system. The purpose of the present study was to investigate the underlying regulatory mechanisms. Co-cultivation experiments between Con A-stimulated OS and NWL lymphocytes in communicating cultures revealed soluble regulatory factors to be responsible for the observed functional differences: the high proliferative response to Con A and hyperproduction of IL 2 of OS cells was found to be due to a deficiency in the conditioned medium of dialyzable inhibitory factor(s) that regulate IL 2 secretion of NWL lymphocytes. Furthermore, sera of young NWL chickens were found to profoundly inhibit the IL 2-promoted lymphoblast proliferation. This IL 2 antagonizing activity is lost with age (3 to 6 yr) and was found to be significantly diminished in OS birds throughout ontogeny, thus pointing to possible parallels between immune regulatory dysfunction in autoimmunity and in physiologic aging. Both enhanced T cell response and the defect in serum suppressor were inherited by (OS X CB)F1 animals, indicating that these two aberrations may be related to each other.

Thyroid glands of Obese strain (OS) chickens with spontaneous hereditary autoimmune thyroiditis were studied by direct immunofluorescence (DIF) with an anti-chicken immunoglobulin–FITC conjugate for local immunoglobulin (Ig) production in plasma cells and germinal centres. Many plasma cells and most of the germinal centres showed positive staining in DIF. The Ig property of this stained material was verified by DIF blocking and indirect immunofluorescence (IIF) tests with specific unlabelled anti-chicken Ig sera. In a chronological DIF study of thyroid glands from OS chickens aged 1–18 weeks, Ig-producing plasma cells could be already detected in the 1st week of life. DIF tests with TRITC-labelled chicken thyroglobulin revealed positive staining of plasma cells with preferential localization in close proximity to, or even between, follicular epithelial cells, suggesting the anti-thyroglobulin autoantibody nature of at least some of the locally produced Ig.